CS203159B2 - Process for preparing antibiotic l 13 365 - Google Patents

Abstract

A biologically pure culture of Actinoplanes sarveparensis.

Description

The invention relates to a process for the production of a novel antibiotic obtained by fermentation of a strain belonging to the genus Actinoplanes named Actinoplanes sarveparensis. The novel antibiotic referred to herein as the L 13 365 antibiotic is a pale yellow crystalline substance, possessing characteristics such as melting point, infrared and ultraviolet absorption maxima.

As mentioned above, the L 13 365 antibiotic is produced by fermentation of the Actinoplaines sarveparensis strain. This strain is identified by its collection number A / 13 826 and was isolated from a soil sample taken in the village of Servepar (India). The tribe was deposited and became part of the stock collection of CBS cultures (Central Bureau Voor Schimmelcultures - Oosterstraat 1 - Baarn — the Netherlands), where it was designated 350.76.

To prepare the L 13 365 antibiotic, the microorganism is cultured under aerobic conditions in an aqueous nutrient medium containing assimilatable carbon sources, assimilatable nitrogen sources, and inorganic salts.

Typically, the antibiotic-producing strain is first cultured in a shake flask until sufficient antibiotic activity is present, then the culture is used to inoculate industrial fermenters containing a nutrient fermentation medium.

SUMMARY OF THE INVENTION The present invention provides a method for producing an L 13 '365 antibiotic, characterized in that Actinoplanes serveparensis CBS No. 305.76 is grown in a culture medium comprising a usable carbon source, a usable nitrogen source and an inorganic salt in an aerobic suibmer culture at at 25-35 ^Q C, pM 6-10, for 72 to 96 hours in the accumulation of the antibiotic in culture prcistředí, whereupon the antibiotic from the filtered broth is isolated with an organic solvent and an alkanol of 1 to 6 carbon atoms, alkyl lower aliphatic acids having 1 to 4 carbon atoms in the alkyl ester moiety or lower halogenated hydrocarbons. During cultivation, microbiological tests are performed by diffusion method on agar to determine the concentration of antibiotic produced.

After removal of the mycelliferous sludge by filtration, an antibiotic is recovered from the profitable fermentation solution by conventional methods, such as extraction with an organic solvent in which the antibiotic is soluble and which is immiscible with the aqueous medium. Extraction is carried out after adjusting the pH of the filtrate to 2-4. Suitable organic solvents for the extraction are preferably selected from alkanols containing 1 to 6 carbon atoms, (C1-C6 alkyl esters of lower aliphatic acids, or lower halogenated hydrocarbons). is then separated from the fermentation solution by high speed centrifugation, concentrated to 1/200 to 1/400 of its original volume, cooled and allowed to stand until a prepicitate is formed which is separated by filtration. 13 365.

The mother liquors are collected and poured in excess into an inert nonpolar solvent such as light kerosene to provide additional crude antibiotic L 13 365. The crude product thus obtained is dissolved in a mixture of methanol and acetone and precipitated from the solution by the addition of acidified water.

The two portions were combined and further purified by column chromatography using a mixture of chloroform and methanol as eluent.

The L 13 365 antibiotic is finally crystallized from a 1: 1 mixture of methanol and ethyl acetate.

The antibiotic L 13 365 shows considerable antibacterial activity both in vitro and in vivo. In particular, the antibiotic L 13 365 shows an excellent antimicrobial action in vitro especially against gram positive bacteria, as shown in the following table:

Table

Strain Minimum inhibitory concentration [µg / ml]

Staphylococcus aureus

ATCC 65138 0.025

Staphylococus aurees Tour 0.005 '

Stre, Ptococcus haemolyticus

C 203 0.0062

Strain Minimum inhibitory concentration (µg / jnl)

Diplococcus pneumoniae

UC 41 0.00078

Clostridium perfringens

ISS 30543 0.012

Mycoplasma gallisepticum

H21 C.Z.B. 0.2

In addition, as noted above, the L 13 365 antibiotic also exhibits excellent in vivo efficacy against experimental infections in mice. The table below shows the mean effective doses (EDsoj antibiotic L 13 365 against experimental infections caused by Staphylococcus aureus, Streptocooc® haemolyticus and D-iplococcus pneumoniae).

Table

Infectious strain ED50 mg / kg s. C.

Streptococcus ^ι haemuly't'i'Cus0,2

Stapihylococcus aureus10,0

Dljplococeus pneumuniae0,4

The novel antibiotic is also effective against strains of microorganisms that are resistant to commonly used antibiotics. As selected examples, the following Table I lists the minimum inhibitory concentrations (I.I.I.J. of the antibiotic L 13 365 against Staphyl ^ aurus strains resistant to certain antibiotics).

Table I Strain Minimum inhibitory concentration of other antibiotics Minimum inhibitory concentration of antibiotic L 13 365 Penicillin resistant Staphyloeeueus aureus ATCC 6538 penicillin> 100 0.4. Stiaphyllococcus aureus Tour streptomycin resistant streptomycin> 100 0,078 Staphyloeoecss aureus ATCC 6538 teteaeykiins resistant tetracycline> 1-00 0.15 Staphylococcus aureus ATCC 6538 resistant to rifampicin firampicin> 100 0? 01 Staphyloeoecss aureus ATCC 6538 neomycin resistant neomycin> 100 0.005 Stapnylococcus aureus Erythromycin resistant ATCC 6538 erythromycin> 100 0.04

Minimum inhibitory concentration of antibiotic

OJ L 13 365

Strain

Minimum inhibitory concentration of other antibiotics

Staphylococcus aureus ATCIC 6538 resistant to chlorine amphenicol

Staphylococcus aureus

ATOC 6538 resistant to cephalidine

Staphylococcus aureus

ATOC 6538 kanamycin resistant

Staphylococcus aureus

ATOC 6538 bacitracin resistant

Lincomycin resistant Staphylococcus aureus ATOC 6538 chloramphenicol> 1000.02 ceiplialoridiin> 1000.078 kanamycin> 1000.04 ba-citiraioin> 1000.02 lincomycin> 1000.15

The toxicity of the L 13 365 antibiotic when administered orally or intraperitoneally in mice is very low because the mean lethal dose (LD 50) is greater than 1000 mg / kg.

Description Actinoplanes sarveparensis' A / 13 826

Macroscopic examination of colonies

The strain grows well on various nutrient agar. On oatmeal agar, colonies have a diameter of 3 to 4 mm, irregular margins and a rough surface. Airy mycelium is always missing.

Microscopic examination

Sporangia are formed in several agar culture media. They have a regular spherical shape with a diameter of 13 to 20 μ. Zoospores, highly mobile, are spherical but occasionally slightly elongated in diameter

1.5 to 2 μ.

Based on these properties, the A / 13 826 strain is assigned to the genus Actinoplanes and named Actionplanes sarveparensis C. B. S. 305.76. Table I shows cultures of Actinoplanes serveparensis C. B. S. 305.76 grown on various standard formulations designed by Shirling and Gottlieg [Intern. J. Syst. Bact. 16, 313-340, 1966) and in other environments designed by Waksman [The Actinomycetes, Vol. II, The Williams and Wilkins Co., 1961). The characteristics of the cultures were determined after 6-14 days of incubation at 30 ° C.

Table III shows the utilization of carbon sources investigated by the Pridha.mia and G-ottlieib methods (Intern. J. Syst. Bact., 56, 107, 1948).

Table IV lists the physiological properties of the strain.

Actinoplanes sarveparensis C. B. S. 305.76

Culture environment

Culture medium No. 2 (yeast extract— —maltose agar)

Culture medium No. 3 (oatmeal agar)

Culture medium No 4 (inorganic salts - - starch agar)

Culture medium No. 5 (glycerol - asparagine agar)

Culture medium No. 6 (peptone yeast extract - iron agar)

Culture medium # 7 (tyrosine agar)

Oatmeal Agar (Waksman)

Hyckney and Tresner agar

Czapek's glucose agar

Glucose and asparagine agar

Nutrient agar

Potato agar

Bennet agar

Calcium maleate agar

Skim's Milk Agar

Czapkův agar

Egg agar

Agar with peptone and glucose

Agar

Loeffler's serum

Potatoes

Gelatine

Cellulose

Table II Culture characteristics

Characteristics of cultures

Rich growth, wrinkled surface, orange to brown (the central part of the colony is darker).

Slight, thin, glassy to pale orange. Sporangia production.

Medium size, crust, light orange (9 / G / 7).

Rich growth, crusty surface, bright orange (9/1/11).

Slight growth, crusty brown (13 / C / 7).

Medium size, crust, orange (II / F / 8).

Rich, rugged, orange (9 / / H / 12), central. part of the colony is darker.

Medium size, wrinkled surface, brown (18 / A / 12).

Rich, creased, orange (11 / G / H).

Rich growth, rough surface, bright orange (9/1/11).

Medium size, rough surface, orange (II / F / 8).

Medium size, wrinkled surface, orange to brown, sporangia production.

Medium size, wrinkled surface, light orange (9 / D / 9).

Slight growth, mild glassy to pale orange, sporangia production.

Rich growth, wrinkled surface, orange to light brown, speckled shadows.

Rich, creased, orange (11 / G / H).

Slight growth, rough surface, glassy.

Medium size, wrinkled surface, dark orange (11- / B / 12).

Very slight growth, thin, glassy.

Very slight growth, orange.

Slight growth, wrinkled surface, dark orange.

Slight growth, light orange.

No growth.

The colors were determined according to the method of Maerz and Paul (Maerz A. and Paul M. R., 1950). A dictionary of color, 2nd ed. M. Grow — Hill Book Company, Inc., New York).

Some culture medium numbers correspond to those of Shirling and Gottlieb (Intern. J. Syst. Bact., 16, 313-340, 1966).

Table III

Utilization of carbon compounds

Carbon source

Use

C5 á'rabinčza + xylose + c ₆ glucose + frúktóza + mannosis + mannitol inositol rhamnóza + (C6) 2 sucrose + lactose + (Ce) 3 raffinose - (C6) 4 cellulose - Special salicin

+ indicates use - indicates non-use

Table IV

Physiological properties

Test Results starch hydrolysis positive H2S formation positive formation of melanin negative tyrosine reaction negative hydrolysis of casein positive hydrolysis of maleic acid positive calcium nitrate reduction positive coagulation of litmus negative of milk peptonization of litmus positive of milk liquefaction of gelatin positive

Production and isolation of antibiotics

To produce an antibiotic, the Actinoplanes sarveparensis C. B. S. 305.76 strain is cultured under aerobic conditions in the culture medium until sufficient antibiotic activity is found at pH values ranging from 6 to 10. For example, the shake flask culture may have the following composition in g / liter:

meat extract3,0 trypton5,0 yeast extract5,0 glucose1,0 soluble starch24,0 calcium carbonate · calcium distilled water q. up to 1000.0 ml to 30 ° C and pre-cultures (one liter) are used to inoculate industrial fermenters containing 10 liters of the following medium:

meat extract 40.0 0 peptone 40.0 ô yeast extract 10.0 g sodium chloride 25.5 0 soy flour 101.0 0 glucose 500.0 0 calcium carbonate 50.0 0 tap water q. up to · 10 liters

The fermentation batches are incubated under aerobic conditions and agitation at 28 to 30 degrees Celsius. At regular intervals, antibiotic activity is determined microbiologically by agar diffusion using the Staphylococcus aureus test organism. Maximum efficiency is achieved after 72 to 96 hours of fermentation.

Isolation and cleaning of antibiotics L 13 365.

The contents of the fermentation tanks (80 liters) are filtered using ¹ filter aid "1% (w / v) clarcel". The mycell cake was removed and the filtered solution acidified to pH 2.5 with 10% HCl was extracted twice with ethyl acetate corresponding to 50% by volume.

The organic phase was separated from the aqueous using high speed centrifugation, then dried over Na 2 SO 4, concentrated at 45-50 degrees Celsius in vacuo to 1/300 of its original volume, and finally cooled to up to 10 ° C. A crude precipitate is formed, which is obtained by filtration, washed with small

The bottles were shaken for about 24 hours, at about 28 amounts of ethyl acetate, and dried at 45 ° C under vacuum. It is obtained. 1.140 g antibiotic about Í70 why, purity determined spectrophotometrically.

The mother liquors resulting from the above filtration are poured into 20 volumes of light kerosene to give an additional amount of 2.250 g of antibiotic having a purity of 20-25% (determined spectrophotometrically). This substance is suspended in a small amount of methanol / acetone (9: 1), filtered off from an insoluble fraction and dissolved in water: bring the solution to pH

2.5 with 10% HCl with stirring and the precipitate obtained was collected by centrifugation and further dissolved in a minimum amount of butanol at 45-50 ° C. Concentrate the solution in vacuo to 1/5 of the original volume and cool to 4 ° C; the precipitate obtained is collected and dried under vacuum and is an antibiotic L 13 365 (0.450 g) having a degree of purity of 80 why (determined spectrophotometrically). The yields obtained are then subjected to conventional purification procedures. To this end, they are dissolved in a minimum amount of a 85: 15 mixture of chloroform and methanol and chromatographed on a 1: 1 by volume mixture of silica and celite. The column is pre-activated at 100 ° C and washed with the above chloroform / methanol mixture. .

Elution and thin-layer chromatography control of the fractions was performed with the same mixture. Fractions were collected according to thin layer chromatography data and concentrated in vacuo to a small volume. Upon addition of diethyl ether, a precipitate is formed consisting of the antibiotic L 13 365 with a degree of purity of 95% (determined spectrophotometrically). The precipitate is dissolved in a 1: 1 mixture of methanol and ethyl acetate, heated to 45 ° C, filtered from insoluble material. After cooling to 4 ° C and standing overnight at this temperature, pure L 13 365 was obtained as light yellow needles.

Chemical - physical properties of antibiotic L 13 365.

Antibiotic L 13 365 is a pale yellow crystalline powder of acid character. Analysis of the acid hydrolyzate antibiotic L 13 365 in 6 N hydrochloric acid in a sealed tube at 110 ° C reveals four amino acids. Three of them were identified by an automatic analysis instrument such as aspartic acid, threonic acid and glycine. In addition, the L 13 365 antibiotic is characterized by the following properties:

1) Melting point 210 ° C.

2) Elementary analysis:

% C, 51.35;% H, 5.05;% N, 10.50.

11,05% S.

O (from difference): 22.05

3) Ultraviolet and visible absorption, solvent spectrum A _max (nm) 1%

E 1 cm

hydrochloric acid 0,1 N 360 250 (hump) 103 237 326 0.15 M buffer 408 (hump) pH 7.5 378 67 250 (hump) 237 326 0.15 M buffer 408 5Θ pH 8.8 250 (hump) 238 336

The ultraviolet absorption spectra were determined using a UNICAM Sp 800 ultraviolet spectrophotometer using a L 13 365 solution in methylcellosolv (MCS) buffers at different rates. pH in 1: 1 ratio.

The complete waveform is shown in Figure 1.

4) · Fluorescence spectrum

The antibiotic contains a strongly fluorescent chromophore and a solution of the product in 0.1 N sodium hydroxide excited at 240 nm shows an emission spectrum with peaks at 355 and 490 nm.

The fluorescence spectrum was determined with a Perkin fluorescence spectrophotometer

Elmer Mod MPF — 44.

5) Infrared spectrum

The characteristic absorption bundles in nujol were observed at the following frequencies (cm -1): 3400 · (hump), 3350 (sharp), 3200 (hump), 3100 (sharp), 2930 and 2870 (nujol), 2800—2400, 2380 ( atmospheric carbon dioxide), 1750 (sharp), 1670 (sharp), 1620 (sharp), .1540 (sharp), 1480 (sharp,

1460 and 1380 (nujol), 1420 (sharp), 1340 (sharp), 1320 (sharp), 1280 (n), 1240

(. sharp), 1195 (sharp), 1170 (sharp ,, 1145 (sharp), 1120 (. sharp) »1075 (wide), 1015 (sharp), 550 (sharp), 535 (sharp), 520 (wide) , 500 · (sharp), 865 (sharp), 840 (-sharp),

800 (sharp), 770 (sharp), 755 (sharp), 740 (sharp), 720 (nujol).

The infrared spectrum was determined on a Perkin-Elmer Mod 157 spectrophotometer.

The complete spectrum image is shown in Figure 2.

6) Specific rotation [w] 436 ²⁵ - + 125 ° (C - 0,8% in methanol / chloroform 1: 1)

7) Solubility

The compound is soluble in dimethylsulfoxide, dimethylformmiamide, and chloroform-methanol mixtures, sparingly soluble in chloroform, methanol, methanol / ethyl acetate mixtures, in sodium bicarbonate and glacial acetic acid solutions, insoluble in water and in other common organic solvents.

8) Characteristic reactions

Fehling

Tollens

KMnO4 H2SO4 conc. ninhydrin

FeCl3

Milloinova

Schiffova

Anthroin

Folin Ciocalteau Elson Morgan positive positive positive dark brown color negative positive negative negative positive negative negative

9) Acidity

An ionizable function with a pKa of 7.8 is shown by potentiometric titration with 0.1 N sodium hydroxide of L 13 365 antibiotic in methylcellosolv: water 4: 1. The equivalent determined correspondingly is 1400.

10) Chromatographic properties

Solvent system

Buffer pH 6.0 saturated with n-butanol 0.75 Water saturated with n-butanol and 2% p-toluenesulfonic acid 0.80

Water saturated with n-butanol and 2% concentrated ammonia 0.70

Ammonium chloride (20% w / v in water)

Butanol saturated buffer pH 6.00.0 n-butanol: methanol: water 40: 10: 20 containing 0.75 g of methylene chloride 0.90 n-butanol: methanol: water 40: 10: 300.90 water: acetone 1:: 10.35 water saturated with ethyl acetate 0.0-0.31 chloroform: methanol 85:15 (xx) 0.51 < ^x > Paper chromatography on Whatman # 1. The antibiotic was visualized on agar plates inoculated with S. aureus.

(xx) Thin-layer chromatography on silica gel plates MF / UV254. Fluorescent spot under ultraviolet light.

Claims (1)

OBJECT OF THE INVENTION Process for the production of the antibiotic L 13 365, characterized in that the Actinoplanes serveparensis C. B.S. No. 305.76 in a culture medium comprising a usable carbon source, a usable nitrogen source, and inorganic salts in a submerged aerobic culture; at a temperature of 25 to 35 ° C at pM of 6 to 10 for 72 to 96 hours until the antibiotic has accumulated in the culture medium, whereupon the antibiotic is isolated from the filtered culture medium with an organic solvent, a C 1 -C 6 alkanol, an alkyl ester aliphatic acids having 1 to 4 carbon atoms in the alkyl ester moiety, or halogenated hydrocarbons.